All cells possess mechanisms to maintain integrity of the cellular genome through detection and repair of, for example, adduct formation, cross-linking, single-strand breaks, and double-strand breaks. The mechanisms of detection and damage repair, collectively, are called DNA repair. DNA repair functions are carried out on lesions that arise from exposure to a variety of environmental chemical and physical agents, as well as from toxic agents generated intracellularly in normal cellular metabolism. Because DNA provides the information required for cell, tissue, and organism function, a large amount of cellular energy is devoted to maintaining intact structure of the genome.
The most genotoxic damages are those which induce DNA chain disruptions, particularly double-strand breaks. DNA double-strand breaks (dsbs) can be induced by chemical or physical agents, including intercalating agents, electrophilic compounds and ionizing radiation. At least two pathways responsible for the repair of DNA dsbs exist, i.e., homologous recombination (HR) and nonhomologous end joining (NHEJ). The former reaction requires undamaged DNA from the homologous chromosome to be used as a template in the repair of the DNA discontinuity. NHEJ, in contrast, is DNA homology independent and simply requires two free DNA ends to be re-ligated. The exact molecular mechanisms by which both HR and NHEJ are effected remain to be elucidated.
DNA dsbs also are generated during the course of normal cellular development in some tissues. This observation first was appreciated following the discovery and characterization of the severe combined immuno-deficiency (scid) mouse. The scid syndrome is a genetic disorder which manifests as an absence of B- and T-cell immunity (Bosma et al., Nature, 301:527–530 (1983), and reviewed in Bosma and Carroll, Annu. Rev. Immunol., 9:323–350 (1991)). The scid mouse is defective in the earliest stages of lymphoid cell development as a result of an inability to correctly rearrange T-cell receptor (TCR) and IgM μ chain DNA (Bosma and Carroll, Annu. Rev. Immunol., 9:323–350 (1991), Dorshkind et al., J. Immunol., 132:1804–1808 (1984), Lauzon et al., J. Exp. Med., 164:1797–1802 (1986), Schuler et al., Cell, 46:963–972 (1986), Tutt et al., J. Immunol., 138:2338–2344 (1987), Lieber et al., Cell, 55:7–16 (1988)). As a result, T- and B-cells do not progress beyond the CD25+ CD4− CD8− and CD25− pro-B cell stages, respectively. Site-specific V(D)J recombination is initiated in scid mice through the activity of the RAG1 and RAG2 gene products, however, resolution of recombination intermediates is disrupted (Fulop and Phillips, Nature, 347:479–482 (1990), Biedermann et al., Proc. Natl. Acad. Sci. USA, 88:1394–1397 (1991), Hendrickson et al., Proc. Natl. Acad. Sci. USA, 88:4061–4065 (1991), Oettinger et al., Science, 248:1517–1522 (1990), Mombaerts et al., Cell, 68:869–877 (1992), Shinkai et al., Cell, 68:855–867 (1992), van Gent et al., Cell, 81:925–934 (1995), and reviewed in Lieber, FASEB J., 5:2934–2944 (1991)). Nonproductive rearrangements in scid cells typically result in large deletions at the TCR and Ig loci, while the processing of recombination signal sequences is not affected in these cells. The scid mutation, therefore, specifically disrupts the formation of recombinant coding junctions (Lieber et al., Cell, 55:7–16 (1988), Malynn et al., Cell, 54:453–460 (1988)).
The defect in the scid mouse is caused by mutation of the gene encoding the catalytic subunit of the DNA-dependent protein kinase (DNA-PK) (Blunt et al., Cell, 80:813–823 (1995), Peterson et al., Proc. Natl. Acad. Sci. USA, 92:3171–3174 (1995)). Specifically, a nonsense mutation at tyrosine-4046 results in the deletion of the last 83 amino acid residues (Blunt et al., Proc. Natl. Acad. Sci. USA, 93:10285–10290 (1996), Danska et al., Mol. Cell. Biol., 16:5507–5517 (1996), Araki et al., Proc. Natl. Acad. Sci. USA, 94:2438–2443 (1997)).
DNA-PK is a trimeric complex composed of a p460 catalytic subunit and Ku80 (86 kDa) and Ku70 regulatory proteins. Ku70 and Ku80 were initially described as human autoantigens and function as cofactors in vitro stimulating protein kinase activity through binding DNA (Mimori, J. Clin. Invest., 68:611–620 (1981), Dvir et al., Proc. Natl. Acad. Sci. USA, 89:11920–11924 (1992), Gottlieb and Jackson, Cell, 72:131–142 (1993)). Ku70 and Ku80 exhibit highest affinity for DNA duplex termini and gaps (Blier et al., J. Biol. Chem., 268:7594–7601 (1993), Falzon et al., J. Biol. Chem., 268: 10546–10552 (1993)). Although, the precise function of DNA-PK and its natural substrates remain unknown, this enzyme phosphorylates a number of proteins in vitro, including many transcription factors and p53 (Lees-Miller et al., Mol. Cell. Biol., 12:5041–5049 (1992), Anderson and Lees-Miller, Crit. Rev. Euk. Gene Exp., 2:283–314 (1992)).
Cultured scid cells are sensitive to killing by agents that induce DNA double-strand breaks (dsbs), indicating a role for DNA-PK in the repair of these lesions. The scid defect also sensitizes mice to radiation-induced lymphomagenesis (Lieberman et al., J. Exp. Med., 176:399–405 (1992)). Lymphomas arise in scid mice at frequencies ranging from 50 to 100% at x-ray doses that do not affect wild-type mice. Since unirradiated scid mice are not particularly sensitive to lymphomagenesis, the background level of tumor-inducing dsbs must either be low enough to be effectively repaired or the damaged cells are effectively eliminated.
The therapeutic benefit of radiation and chemotherapy in the treatment of cancer is well documented. These physical and chemical agents act by disrupting DNA metabolism at the level of DNA structure, synthesis, transcription and chromosome transmission. Most of these agents act by inducing DNA-specific lesions. Presumably, if tumor cells are sensitive to therapies that introduce DNA specific lesions, then these therapies will be made more effective by simultaneously disrupting the cellular repair of these damages. Therefore, inhibition of cellular DNA-PK activity following treatment with agents that induces DNA dsbs will potentiate the therapeutic index of these agents.
Thus, there exists a need in the art to identify compounds that can improve the efficiency of radiation and chemotherapy in treatment of cancer. Identification of DNA-PK inhibitors can permit development of treatment regimens that include lower doses of radiation and/or chemotherapy drugs, thereby reducing the unwanted side effects that often accompany the treatments.